The invention is in the field of adenoviral vectors and their use in treating disease.
Basic Adenoviral Vector Technology
Adenoviruses (Ad) consist of nonenveloped icosahedral (20 facets and 12 vertices) protein capsids with a diameter of 60-90 nm and inner DNA/protein cores (Horwitz, 1990). The outer capsid is composed of 252 capsomers arranged geometrically to form 240 hexons (12 hexons per facet) and 12 penton bases; the latter are located at each vertex from which protrude the antennalike fibers. This structure is responsible for attachment of Ad to cells during infection. Wild-type Ad contain 87% protein and 13% DNA and have a density of 1.34% g/ml in CsCl.
The double-stranded linear DNA genome of Ad is approximately 36 kb, and is conventionally divided into 100 map units (mu). Each end of the viral genome has a 100-150 bp repeated DNA sequence, called the inverted terminal repeats (ITR). The left end (194-385 bp) contains the signal for encapsidation (packaging signal). Both the ITRs and the packaging signal are cis-acting elements necessary for adenoviral DNA replication and packaging (Sussenbach, 1984; Philipson, 1984).
A simplified map of the adenovirus type 5 (Ad5) genome with a few key landmarks is diagrammed in FIG. 1 (Stratford-Perricaudet and Perricaudet, 1991; Graham and Prevec, 1991). The early (E) and late (L) regions of the genome contain several transcription units and are divided according to the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome as well as a few cellular genes (Nevins, 1993). The expression of the E2 region (E2A and E2B) leads to the synthesis of the proteins needed for viral DNA replication (Pettersson and Roberts, 1986). The proteins from the E3 region prevent cytolysis by cytotoxic T cells and tumor necrosis factor (Wold and Gooding, 1991). The E4 proteins are involved in DNA replication, late gene expression and splicing, and host cell shut-off (Halbert et al, 1985). The products of the late genes, including the majority of the viral capsid proteins, are expressed after processing of a 20-kb primary transcript driven by the major late promoter (MLP) (Shaw and Ziff, 1980). The MLP (located at 16.8 mu) is particularly efficient during the late phase of infection, and the mRNAs issued from this promoter possess a 5xe2x80x2 tripartite leader (TL) sequence, which increases the preference of the host cell for such transcripts as opposed to host cell mRNAs.
The use of Ad as vectors for expression of heterologous genes began soon after the observation of hybrids between Ad and simian virus 40 (SV40) during the 1960s. Since then, Ad vectors have gradually developed into one of the major viral vectors in the current field of gene therapy, because: (a) Ad have been widely studied and well characterized as a model system for eukaryotic gene regulation, which served as a solid base for vector development; (b) The vectors are easy to generate and manipulate; (c) Ad exhibits a broad host range in vitro and in vivo with high infectivity, including non-dividing cells; (d) Ad particles are relative stable and can be obtained in high titers, e.g., 1010-1012 plaque-forming unit (PFU)/ml; (e) The life cycle of adenovirus does not require integration into the host cell genome, and, therefore, the foreign genes delivered by Ad vectors are expressed episomally, thus having low genotoxicity if applied in vivo; (f) Side effects have not been reported following vaccination of U. S. recruits with wild-type Ad, demonstrating their safety for in vivo gene transfer. Ad vectors have been successfully used in eukaryotic gene expression (Levrero et al, 1991; Ghosh-Choudhury, 186), vaccine development (Granhaus and Horwitz, 1992; Graham and Prevec, 1992), and gene transfer in animal models (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al, 1992; Rich et al, 1993). Experimental routes for administrating recombinant Ad to different tissues in vivo have included intratracheal instillation (Rosenfeld et al, 1992), muscle injection (Quantin et al, 1992), peripheral intravenous injection (Herz and Gerard, 1993), and stereotactic inoculation to brain (LaSalle et al, 1993). The initial Ad-mediated gene therapy trial in humans was the transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to lung tissues (Crystal et al, 1994).
Gene-Transfer Mediated Anticancer Immunity
One of the most effective current approaches to cancer gene therapy involves alteration of the tumor-host relationship and facilitation of recognition and destruction of malignant cells by the host immune system. In the tumor-bearing individual, a lack of an effective immune response may be due in part to either weak tumor cell antigenicity, lack of immune co-stimulation, or a tumor-specific immunosuppressive environment. Gene transfer of cytokines to tumor cells provides a strategy for augmentation of an effective anti-tumor immune response (Miller et al, 1994). In recent years, a number of cytokine genes have been isolated, cloned and characterized. Systemic administration of certain of these immunomodulators, such as IL-2, has resulted in an anti-tumor response. However, significant toxicity has accompanied the use of many of these biologics owing to the high concentrations needed to generate clinical effects. The combination of significant undesired effects and marginal therapeutic outcomes from systemic administration has stimulated efforts to genetically engineer tumor cells to produce the cytokines themselves (Rosenberg et al, 1989).
In animal models, gene-modified tumor cells have been used as vaccines to stimulate anti-tumor response (Miller et al, 1994; Dranoff and Mulligan, 1995). The appeal of tumor directed cytokine gene transfer is that the cytokine, produced locally, is immunologically more efficient and does not cause systemic toxicity. Tumor antigens expressed on neoplastic cells in combination with high local concentrations of cytokine(s), creates an immunological micro-environment virtually impossible to reproduce with exogenous cytokine administration. This immunological micro-environment created by such cytokine-producing tumor cells has been shown to result in generation of cytotoxic T lymphocytes. In a number of different animal models, cytokine-producing tumor cells have been shown to be effective in decreasing the tumorgenicity and increasing the expression of immunologically important molecules (Miller et al, 1994; Dranoff and Mulligan, 1995). The initial antitumor rejection appears to be accompanied by a nonspecific inflammatory response. However, rejection of cytokine secreting tumor cells has in most instances led to the generation of systemic, tumor specific immunity that is T cell-dependent.
In addition, new evidence indicates that co-stimulation of T cells by the co-stimulatory molecule B7 has both a positive and negative effect on T cell activation (Leach et al, 1996). Other co-stimulatory molecules for T cells such as ICAM-I, LFA-3 and VCAM-I have also been implicated in the induction of an anti-tumor response (Springer, 1990). The most powerful of these co-stimulatory signals is provided by the interaction of CD28 on a T cell with either or both of its primary ligands, B7-1 (CD80) and B7-2 (CD86) on the surface of an antigen presenting cell (Lenschow et al, 1996). In a variety of model systems, tumor cells transfected with the B7 cDNA induced potent antitumor responses against both modified and unmodified tumor cells (Townsend and Allison, 1993)
It has long been known that both Class I and Class II MHC molecules are involved in the tumor antigen presentation, although different pathways are utilized by the two classes of molecules. Class I MHC has been shown to activate tumor-specific CTL in vitro. Early work on tumor vaccination included transfection of MHC class I genes and resulted in suppression of the tumor cells in tumorigenicity and/or metastasis in mouse models (Hui et al, 1984; Wallich et al, 1985) MHC class II genes were shown to be involved in activation of tumor-specific T-helper cells, and the introduction of Class II genes into tumor cells resulted in a decrease in the tumorigenicity and generated a systemic immune response against the parental tumor (Ostrand-Rosenberg, 1990). Despite these positive results, the relationship between levels of MHC expression and immunogenicity is inconsistent among tumor models. Researchers have recently begun to believe that the inconsistency is caused by other cofactors, such as the B7 co-stimulatory molecule, which affects the antigen presentation by MHC/peptide complexes.
Interferon gamma (IFN-xcex3) is a pleiotropic cytokine that, for example, activates macrophages and plays an important role in the inflammatory response (Billiau, 1996). This pleiotropic cytokine is also a potent inducer of MHC class I and class II antigens and thus is capable of enhancing immune responses (Wallach et al, 1982; Chen et al, 1986). Retroviral transduction of a cDNA encoding murine IFN-xcex3 into a non-immunogenic murine sarcoma cell line that expresses low levels of MHC Class I only weakly induced upregulation of MHC class I antigen expression and generated anti-tumor CD8+ TIL. Following tumor rejection, long-lasting protection from rechallenge with parental cells was induced (Nicholas et al, 1992). Moreover, innoculation of mice having micro-metastases with tumor cells producing large amounts of IFN-xcex3 almost completely cured these mice by inducing CTL (Porgador et al, 1993). The cDNA for human IFN-xcex3 has also been introduced into human renal cancer cells and melanoma cells (Gansbacher et al, 1992; Gastl et al, 1992). Renal cancer cells secreting IFN-xcex3 showed increased expression of MHC class I antigen, xcexc2-microglobulin, and intracellular adhesion molecule I, as well as induction of MHC class II antigen expression. However, tumor formation by a human renal cancer cell line transplanted into nu/nu mice was not affected by IFN-xcex3 secretion, whereas IL-2 production inhibited growth of the tumor.
Many other cytokines, chemokines, and intercrines have been shown to play several different roles in eliciting antitumor immunity (Allione et al., 1994; Plata-Salaman and Borkoski, 1994; Zhang and Fang, 1995). Further studies using gene transfer of multiple cytokine or immuno-stimulatory genes have obtained induction of stronger anticancer immunity (Vagliani et al, 1996). The cytokine or immune modulatory genes have also been used in combination with other gene transfer for development of more effective approaches to gene therapy of cancer (Zhang and Fang, 1995).
Genetically-Engineered Virus Therapy of Cancer
Since the 1920s, viruses have been utilized to induce oncolysis (See review article: Sinkovics and Horvath, 1993). Occasionally, natural human viral infections induce remissions of leukemia or lymphomas. Inoculation of tumor-bearing patients with live viruses were initiated in the late 1940s. Early studies utilized attenuated liver rabies vaccine to treat melanoma, and induced partial remissions. This was followed by clinical trials to measure the effect of myxo-, paramyxo- and arboviruses on various malignancies. Occasional temporary regressions of tumors were observed, however, regression was eventually followed by regrowth of tumor and death of patients. Other investigators used lymphocytopenic murine virus, live mumps virus or human enteroviruses to treat cancers through various routes including intra-vein, intra-tumor, ingestion, or inhalation, but did not obtained documented cases of cancer cures (Sinkovics and Horvath, 1993).
Parvoviruses are small single-stranded DNA viruses which replicate in the nucleus and are able to infect insects, birds, and a variety of mammals, including humans. The parvoviruses of vertebrates are divided into the groups adeno-associated viruses (AAV) and autonomous parvoviruses on the basis of the requirement of the former for helper viruses such as Ad. The distinction, however, is not absolute. Parvovirus replication depends on host cell factors, some of which are expressed during cell proliferation and differentiation (Cotmore and Tattersall, 1987). The outcome of parvovirus infection is dependent on the physiological state of the host cells. Recent studies have shown that a number of human and murine cells committed to neoplastic transformation are significantly more sensitive to the killing effect of prototype strain MVMp of autonomous parvovirus minute virus of mice (MVM) or H-1 than are their normal progenitors (Cornelis et al, 1988; Cornelis et al, 1990; Spegelaere et al, 1991).
Although the molecular mechanism underlying modulation of autonomous parvovirus-host cell interactions by neoplastic transformation is poorly understood, the potential application of the transformation-dependent replication of the viruses has been considered for development of recombinant vectors for tumor-specific killing (Russell et al, 1992; Dupont et al, 1994). However, preliminary experimental results showed that an additional mechanism is needed for the viral vector to induce effective transformed-cell killing (Personal communication with Dr. Francis Dupont).
Wild-type live human adenoviruses has been utilized to treat cervical cancer (Smith et al, 1956). Large amounts of virus were given to patients through intratumoral, intra-arterial, or intravenous inoculation. An appreciable illness or change other than necrosis of cancer tissue was not observed. The autopsy findings confirmed the clinical observations that the injected virus produced only local effects on the cervical tumor and did not affect the progressive growth of tumor in the pelvic tissues or the development and growth of metastases.
The use of Ad vectors in gene therapy have been rapidly developed. The application potential of the genetically-engineered Ad for gene therapy of cancer has been widely explored with many different strategies (Descamps et al, 1996). Ad vector-mediated delivery of genes encoding such proteins as cytokines, interferons, co-stimulatory molecules or factors have induced anticancer immunity in various animal models (Addison et al., 1995; Zhang et al., 1996). However, the efficacy of this type of approach in treatment of human cancer remains to be determined through clinical trials.
Utilization of Tumor-specific or Tissue-specific Promoter/Enhancer Cassettes
Both immuno-gene therapy and virus-mediated gene therapy for cancer have limitations in either therapeutic efficacy or specificity in cancer cell killing. For the latter, novel approaches have been proposed and tested with the goal of targeting gene expression specifically to tumor cells. Progress has been made in targeting infectious recombinant viral vectors to cells through cellular surface receptors by genetic or biochemical modification of the viral surface. An alternative approach is to target cancer cells at the transcriptional level using lineage-specific promoters that restrict expression of effector genes to tumor cells and related normal cells derived from the same developmental lineage (Hart, 1996). Examples of tumor types that have been targeted in this manner include colon (Osaki et al., 1994; Richards et al., 1995), lung (Osaki et al., 1994; Smith et al., 1994), breast (Manome et al., 1994), hepatocellular carcinomas (Huber et al, 1991; Ido et al, 1995; Kaneko et al, 1995), and melanoma (Vile et al, 1993; Siders et al, 1996).
The application of tumor- or tissue-specific promoter/enhancers has also been used in a therapeutic approach called xe2x80x9cvirus-directed enzyme/prodrug therapyxe2x80x9d (VDEPT) (Huber et al, 1991). These studies demonstrated enhancement of tumor-killing efficacy and reduction of the side effects of such therapy on normal cells (the xe2x80x9cbystander effectxe2x80x9d) by tissue- or tumor-specific driven expression of prodrug-activation genes (Manome et al, 1994; DiMaio et al, 1994).
The xcex1-fetoprotein (AFP) promoter/enhancer cassettes have been utilized to control E1 expression from an Ad vector in order to induce a virus-mediated oncolytic effect on hepatocellular carcinoma (Hallenbeck et al, 1996). As proof of concept for the first generation of a tumor specific replication competent adenoviral (TSRCA) vector, the Ad5 E1 promoter of a wild-type Ad was replaced with a modified version of the AFP promoter. The vectors were shown to replicate in two-thirds of human hepatocellular carcinoma cell lines tested that expressed high levels of AFP. Furthermore, approximately 500-1000 hepatocellular carcinoma cells per virion particle were destroyed in a 13 day assay. Little to no replication was observed in two liver cell lines, two lung cancer cell lines, one colon cancer cell line, and one cervical cancer cell line, each of which do not produce AFP. In addition, investigators tested two primary cultures of normal human lung epithelial and endothelial cells for replication of the vectors since lung tissue is the primary target for Ad replication in human. Neither primary culture supported replication of the vectors, demonstrating the specificity of the vectors in cancer cell killing (Hallenbeck et al, 1996). The investigators also proposed the use of other tumor-specific promoter/enhancers of different cancers using the same type of design as the TSRCA vector.
Therapy of cancer using wild-type viruses seldom results in durable, complete remission. The use of genetically-engineered viral vectors to deliver a gene encoding a toxin, cytokine, or immuno-stimulatory factor has demonstrated certain encouraging results using animal models. However, clinical application of methods is limited by the inability to specifically target the vectors or gene expression to cancer cells. The TSRCA approach has addressed the tumor-specific killing through replication of Ad vectors in cancer cells, which may induce oncolytic effect. This method is limited because tumor regression is limited to the local environment. Additionally, the capacity for insertion of heterologous DNA into the TSRCA vector is limited to less than 4 kb.
The present invention provides a solution to the limitations of current TSRCA methodologies. The reagents and methodologies of the present invention allow for the development of TSRCA vectors carrying multiple gene expression cassettes encoding cytokines, chemokines, tumor suppressors, and/or immunomodulatory factors. The inventions of the present invention will be appreciated by those skilled in the art to address and provide solutions for these significant limitations.
This invention encompasses a composition for killing target cells, such as tumor cells. The composition comprises a first and a second adenoviral vector that have complementary function and are mutually dependent on each other for replication in a target cell. One of said adenoviral vectors has a target cell-activated promoter or an early gene deletion that controls and limits propagation of the adenoviral vectors in the target cells. One of the_adenoviral vectors comprises partial Ad genome which can support the replication cycle_of Ad in the target cells. The replication of these vectors in the target cells directly or indirectly kills the target cells. The target cells may be hepatoma, breast cancer, melanoma, lung cancer, colon cancer, or prostate cancer cells, for example. The vectors of this invention may also be utilized to treat other diseases such as restenosis, in which case the target cell may be a vascular smooth muscle cell.
The present invention also comprises an adenoviral vector (the xe2x80x9ccontrolled vectorxe2x80x9d) that comprises a promoter that is activated in a tumor cell and is operably linked to the Ad E1 gene. The products of the E1 gene control the replication of the Ad vector in tumor cells. A deletion of the AD E1 gene can also be used to render the controlled-Ad a capability to specifically drive the viral replication in the tumor cells. The controlled vector may also contain a cassette to express an immunomodulatory protein such that the protein is expressed in the tumor cell. The tumor-activated promoter may, for example, be the xcex1-fetoprotein promoter, the DF-3 mucin enhancer, the tyrosinase promoter, the carcinoembryonic (CEA) promoter, the prostate specific antigen (PSA) promoter, or the H1 parvovirus promoter. The immunomodulatory protein may, for example, be an interferon (IFN) such as IFN-xcex3, a B7 co-stimulatory molecule such as B7.1, an interleukin, a chemokine or a tumor-specific antigen. For other applications, the promoter also can be cell cycle specifically inducible or synthetic promoters/enhancers.
This invention also comprises a xe2x80x9csupplemental vectorxe2x80x9d which provides proteins required for replication of the controlled vector and the supplemental vector in the tumor cell.
This invention also includes methods of making and purifying the vectors of the invention by transfecting cell lines in which the vectors can replicate and are packaged in large amounts. Purification may be completed using a biochemical technique such as cesium chloride (CsCl) centrifugation, for example.
The present invention further provides a modified adenoviral vector which: 1.) increases the capacity of the vector for carrying one or multiple therapeutic genes; 2.) targets adenoviral replication to a specific type of host cell or tissue by providing genes (with or without deletion) encoding the AdE1 proteins operably linked to a cell type or tissue-specific transcriptional regulatory region; 3.) induces expression of an immunomodulatory protein or proteins within a tumor cell to increase the immune response against local and distant sites of tumor growth; 4.) provides a composition of tissue specific mutually dependent adenoviral vector hereinafter referred to as xe2x80x9ccomplementary-Ad vector systemxe2x80x9d that induces expression of an immunomodulatory protein and transcriptionally targets adenoviral replication to specific cell types.
It is an objective of the present invention to provide vectors and methodologies needed to combine the specific tumoricidal effect of Ad vectors with induction of systemic anticancer immune responses by delivery of effector genes such as cytokines, chemokines, tumor antigens, MHC molecules, cell adhesion molecules, and/or other immunomodulating factors. Preferably, the present invention provides a controlled-Ad vector having large gene-carrying capacity that may be utilized to deliver single or multiple expression cassettes comprising immunomodulatory or tumor suppressor genes. Ideally, the complementary-Ad vector system induces a local specific-tumoricidal effect together with a systemic anticancer immune response. Therefore, it is further an objective of the present invention to provide specific tumor cell killing at local and remote sites for elimination of primary and metastatic cancer cells.
It is yet another objective of the present invention to provide two Ad vectors that complement each other for replication and have separate roles in lysing tumor cells and delivering effector genes. The present invention therefore provides a supplemental-Ad vector, having an E1 region deletion or substitution and a modified or unmodified packaging signal, and that provides the viral DNA replication and capsid proteins necessary for packaging of the controlled-Ad. In a preferred embodiment the supplemental Ad vector has a modified packaging signal and the controlled-Ad vector has a wild-type packaging signal to gain packaging advantages over the supplemental Ad vector and the E1 region controlled by a tissue-specific or tumor-activated promoter/enhancer cassette for trans-activation of the supplemental-Ad transcription and replication.
It is also an objective of the present invention to provide tumor-directed cytokine gene transfer such that the cytokine is produced locally, thus providing an immunologically more efficient system and does not cause systemic toxicity. The combination of significant undesired effects and marginal therapeutic outcomes from systemic administration has stimulated efforts to genetically engineer tumor cells to produce the cytokines themselves (Rosenberg et al, 1989).
It will be understood by those skilled in the art that the present invention is not limited in application to gene therapy of cancer. Other applications of the present invention are contemplated.